Background: Acute respiratory infection plays an important role in hospitalization of children in developing countries; detection of viral causes in such infections is very important. The respiratory syncytial virus (RSV) is the most common etiological agent of viral lower respiratory tract infection in children, and human metapneumovirus (hMPV) is associated with both upper and lower respiratory tract infections among infants and children. Objectives: This study evaluated the frequency and seasonal prevalence of hMPV and RSV in hospitalized children under the age of five, who were admitted to Aliasghar children’s hospital of Iran University of Medical Sciences from March 2010 until March 2013. Patients and Methods: Nasopharyngeal or throat swabs from 158 hospitalized children with fever and respiratory distress were evaluated for RSV and hMPV RNA by the real-time polymerase chain reaction (PCR) method. Results: Among the 158 children evaluated in this study, 49 individuals (31.1%) had RSV infection while nine individuals (5.7%) had hMPV infection. Five (55.5%) of the hMPV-infected children were male while four (44.5%) were female and 27 (55.2%) of the RSV-infected patients were females and 22 (44.8%) were males. The RSV infections were detected in mainly < one year old children and hMPV infections were detected mainly in > one year old children. Both RSV and hMPV infections had occurred mainly during winter and spring seasons. Conclusions: Respiratory syncytial virus was the major cause of acute respiratory infection in children under one-year of age while human metapneumovirus had a low prevalence in this group. The seasonal occurrence of both viruses was the same. Keywords: Human Metapneumovirus; Respiratory Syncytial Virus; Children, PCR

Background: To develop hepatitis C virus (HCV) vaccine, induction of potent humoral and T cell response against immunogenic targets with conserved region should be achieved. T cell response against NS3 is often associated with complete clearance of the virus. Objectives: Herein, we expressed the truncated form of NS3 in a mammalian cell line and evaluated immune responses of NS3 DNA vaccine in BALB/c. Materials and Methods: The partial length of NS3 gene, which encodes immunogenic epitopes (1095 - 1379 aa), was amplified by reverse transcription-polymerase chain reaction (RT-PCR) on RNA obtained from a patient with HCV, inserted into pcDNA3.1 plasmid using XhoI/HindIII sites, and finally evaluated by restriction analysis and sequencing. After transfection of the recombinant plasmid into HEK293T cells, the NS3 protein expression was confirmed by western blotting. Mice were immunized intra-dermally close to the base of the mice tail with four doses in two-weeks intervals and the immune responses were assessed using total and subtypes of IgG antibody assay, cell proliferation and cytokine assay. Results: The pcDNA3.1 plasmid harboring the coding sequence of NS3 (pc-NS3) was constructed and confirmed with the expected size. Proper expression of the recombinant protein in transfected HEK 293T cells was confirmed using western blotting. The immunization results indicated that pc-NS3 induced significant levels of total antibody, IgG2a subclass antibody, Interferon (IFN)-γ, Interleukin (IL)-4 and proliferation assay compared to the control group (P < 0.05). Conclusions: The pc-NS3 possesses the capacity to express NS3 in the mammalian cell line and demonstrated strong immunogenicity in a murine model. Our primary results demonstrated that the immunogenic truncated region of NS3 could be used as a potential vaccine candidate against hepatitis C. Keywords: Hepatitis C Virus; NS3; Plasmid; Immunization; DNA Vaccine

Hepatitis C virus (HCV) is a major health problem all over the world. Among HCV proteins, nonstructural protein 3 (NS3) is one of the most promising target for anti‐HCV therapy and a candidate for vaccine design. DNA vaccine is an efficient approach to stimulate antigen‐specific immunity but the main problem with that is less immunogenic efficiency in comparison with traditional vaccines. Several approaches have been applied to enhance the immunogenicity of DNA. Recently, bacteria‐derived substances are considered as one of the most attractive adjuvants for vaccines, which among them, Listeriolysin O (LLO) of Listeria monocytogenes is a toxin with an extremely immunogenic feature. We investigated detoxified form of LLO gene as genetic adjuvant to modulate NS3 DNA vaccine potency. Immunogenic truncated NS3 gene sequence of HCV (1095‐1380aa) and detoxified LLO gene region (5‐441aa) were amplified by PCR and cloned into the pcDNA3.1 plasmid separately. The expression of recombinant proteins (pc‐NS3, pLLO) was confirmed in HEK293T cell line by western blotting. BALB/c mice models received three doses of different formula of plasmids in two‐week intervals and two weeks after the final immunization, the immune responses were evaluated by specific total antibody level, lymphocyte proliferation, cytotoxicity, and cytokine levels assays. To evaluate in vivo cytotoxic activity, tumor challenge was performed. The recombinant plasmids were successfully expressed in mammalian cell line, and coadministration of pc‐NS3 with pLLO induced the highest titer of total IgG against NS3 antigen compared with other controls. Determination of IgG subclasses confirmed the efficient increase in mixed responses with Th1 dominancy. Furthermore, significant levels of cytokines (p < .05) and lymphocyte proliferation responses (p < .05) indicated the superiority of this regimen. The findings may have important implication for LLO gene application as genetic adjuvant in immune response against HCV.